Bottom Line:
In an effort to identify the structural factors contributing to the evolutionary diversification of product specificity amongst this group of enzymes, we selected nine CGTases from both mesophilic, thermophilic and hyperthermophilic organisms for comparative product analysis.These enzymes displayed considerable variation regarding thermostability, initial rates, percentage of substrate conversion and ratio of alpha-, beta- and gamma-cyclodextrins formed from starch.Sequence comparison of these CGTases revealed that specific incorporation and/or substitution of amino acids at the substrate binding sites, during the evolutionary progression of these enzymes, resulted in diversification of cyclodextrin product specificity.

ABSTRACTCyclodextrin glucanotransferases (CGTases) have attracted major interest from industry due to their unique capacity of forming large quantities of cyclic alpha-(1,4)-linked oligosaccharides (cyclodextrins) from starch. CGTases produce a mixture of cyclodextrins from starch consisting of 6 (alpha), 7 (beta) and 8 (gamma) glucose units. In an effort to identify the structural factors contributing to the evolutionary diversification of product specificity amongst this group of enzymes, we selected nine CGTases from both mesophilic, thermophilic and hyperthermophilic organisms for comparative product analysis. These enzymes displayed considerable variation regarding thermostability, initial rates, percentage of substrate conversion and ratio of alpha-, beta- and gamma-cyclodextrins formed from starch. Sequence comparison of these CGTases revealed that specific incorporation and/or substitution of amino acids at the substrate binding sites, during the evolutionary progression of these enzymes, resulted in diversification of cyclodextrin product specificity.

Mentions:
Cyclodextrin size specificity The amount of α-, β- and γ-cyclodextrins produced from starch by the CGTases was followed in time (Fig. 4). The four CGTases from Bacillus species were all primary β-cyclodextrin producers, yielding up to 22.6 g β-cyclodextrin per litre (Table 3). BC251 CGTase initially produced β-cyclodextrin at the fastest rate before levelling off after 24 h at 22.6 g/L (Fig. 4 and Table 3). BA2-5a CGTase was the most “specific” β-cyclodextrin producer with 77% β-cyclodextrin being formed (Table 3). Both Tabium and Toruzyme CGTases produced greater initial amounts of α-cyclodextrin compared to CGTases from Bacillus species. These enzymes also had the highest percentage conversion of starch into cyclodextrins along with BNO2 CGTase (Fig. 4 and Table 3). However, after reaching the maximum cyclodextrin yield, a continuous decrease in the amount of α-, β- and γ-cyclodextrins was noted for both Tabium and Toruzyme CGTases. A. gottschalkii CGTase also degraded cyclodextrins in the later stages of the starch incubations. This strong reduction in cyclodextrin yield may be attributed to the elevated hydrolytic rates of these enzymes on starch, providing short oligosaccharides for the breakdown of cyclodextrins in the coupling reaction (Fig. 1). The absolute yield of α-cyclodextrin was greatest for Thermococcus sp. B1001 CGTase (Fig. 4), even though the percentage of α-cyclodextrin was higher for the K. pneumoniae CGTase (Table 3).Fig. 4

Mentions:
Cyclodextrin size specificity The amount of α-, β- and γ-cyclodextrins produced from starch by the CGTases was followed in time (Fig. 4). The four CGTases from Bacillus species were all primary β-cyclodextrin producers, yielding up to 22.6 g β-cyclodextrin per litre (Table 3). BC251 CGTase initially produced β-cyclodextrin at the fastest rate before levelling off after 24 h at 22.6 g/L (Fig. 4 and Table 3). BA2-5a CGTase was the most “specific” β-cyclodextrin producer with 77% β-cyclodextrin being formed (Table 3). Both Tabium and Toruzyme CGTases produced greater initial amounts of α-cyclodextrin compared to CGTases from Bacillus species. These enzymes also had the highest percentage conversion of starch into cyclodextrins along with BNO2 CGTase (Fig. 4 and Table 3). However, after reaching the maximum cyclodextrin yield, a continuous decrease in the amount of α-, β- and γ-cyclodextrins was noted for both Tabium and Toruzyme CGTases. A. gottschalkii CGTase also degraded cyclodextrins in the later stages of the starch incubations. This strong reduction in cyclodextrin yield may be attributed to the elevated hydrolytic rates of these enzymes on starch, providing short oligosaccharides for the breakdown of cyclodextrins in the coupling reaction (Fig. 1). The absolute yield of α-cyclodextrin was greatest for Thermococcus sp. B1001 CGTase (Fig. 4), even though the percentage of α-cyclodextrin was higher for the K. pneumoniae CGTase (Table 3).Fig. 4

Bottom Line:
In an effort to identify the structural factors contributing to the evolutionary diversification of product specificity amongst this group of enzymes, we selected nine CGTases from both mesophilic, thermophilic and hyperthermophilic organisms for comparative product analysis.These enzymes displayed considerable variation regarding thermostability, initial rates, percentage of substrate conversion and ratio of alpha-, beta- and gamma-cyclodextrins formed from starch.Sequence comparison of these CGTases revealed that specific incorporation and/or substitution of amino acids at the substrate binding sites, during the evolutionary progression of these enzymes, resulted in diversification of cyclodextrin product specificity.

ABSTRACTCyclodextrin glucanotransferases (CGTases) have attracted major interest from industry due to their unique capacity of forming large quantities of cyclic alpha-(1,4)-linked oligosaccharides (cyclodextrins) from starch. CGTases produce a mixture of cyclodextrins from starch consisting of 6 (alpha), 7 (beta) and 8 (gamma) glucose units. In an effort to identify the structural factors contributing to the evolutionary diversification of product specificity amongst this group of enzymes, we selected nine CGTases from both mesophilic, thermophilic and hyperthermophilic organisms for comparative product analysis. These enzymes displayed considerable variation regarding thermostability, initial rates, percentage of substrate conversion and ratio of alpha-, beta- and gamma-cyclodextrins formed from starch. Sequence comparison of these CGTases revealed that specific incorporation and/or substitution of amino acids at the substrate binding sites, during the evolutionary progression of these enzymes, resulted in diversification of cyclodextrin product specificity.